The AcroWell 96 filter plate assembly is constructed of a single-piece polypropylene housing with two layers of membrane welded to the bottom of the plate. The two layers include a single sheet of filter membrane and a single sheet of supported Emflon® membrane, hydrophobic polytetrafluoroethylene (PTFE), both welded to the bottom of the plate using a patented sealing process. This process seals both layers of membrane to each of the 96 wells, eliminating the possibility of lateral crosstalk.

Some 96-well filter plate users expressed concern that because there is no physical separation between the wells, sample could flow through the membrane from one well to the next. To address this issue, we designed specific experiments to demonstrate that lateral flow does not occur between the wells even at extended incubations. To verify that well-to-well crosstalk does not occur, either 10 fmol/well Europium Enhancement Solution (PerkinElmer, Inc., Boston, MA, USA) or 400 pg/well fluorescein in water was added to every other well in the AcroWell 96 filter plate with GHP membrane (PN 5020) in a checkerboard pattern. The negative control wells contained either the Enhancement Solution for Eu-containing plates or Water for Fluorescein-containing plates. After the addition of solutions, the plates were incubated for 24 hours at room temperature and read using a VICTOR* Multilabel Counter at either time-resolved fluorescence (340 nm/615 nm, 0.4 msec delay, 0.4 msec window) or fluorescein (485 nm/535 nm, 1.0 s) factory detection settings. The data from the VICTOR was converted into Microsoft* Excel and the values for all 96 wells were plotted.

The data shown in Figure 1 indicates that the AcroWell 96 filter plate has no well-to-well lateral flow. If the lateral flow of label occurred during this extended incubation, then we would expect to see data points that fall between the values shown (upper data points are wells with added label).

Figure 1. AcroWell Lateral Flow Minimization

The current AcroWell 96 filter plate has been most successful for applications that involve the capture and analysis of particulate on the surface of the membrane. Based on the lack of flow direction present in the current design, we do not recommend the AcroWell 96 filter plate be used to collect filtrate when using vacuum filtration.

Another popular and consistent method for the filtration of multiple samples is to use centrifugation. If centrifugation is used, flow direction is not a problem because the centrifugal force effectively directs the filtrate into the receiving plate. To test whether crosstalk occurs during centrifugation using the AcroWell 96 filter plate, we set up a similar checkerboard experiment using centrifugation with 5 fmol/well free Europium or 400 pg/well fluorescein. Instead of an extended incubation, the plates were incubated briefly and placed into a centrifuge (2 minutes at 3000 x g). The resulting filtrate was collected in a receiver plate and analyzed using a VICTOR* Multilabel Counter at time-resolved fluorescence (340 nm/615 nm, 0.4 msec delay, 0.4 msec window) or fluorescein (485 nm/535 nm, 1.0 s) factory detection settings. The data from the VICTOR was converted into MicroSoft* Excel and the values for all 96 wells were plotted.

The data in Figure 2 shows that no crosstalk is seen in the collection plate when using centrifugal filtration. A difference of up to one order of magnitude was measured between adjoining wells without cross contamination.

Figure 2. Centrifugal Crosstalk Minimization

Despite fears to the contrary about the risk of having a continuous sheet welded to the bottom of a filter plate, our data shows conclusively that lateral flow between wells cannot be detected with AcroWell 96 filter plates. This is seen even when high concentrations of label are added to every other well. In fact, it should be noted for both time-resolved fluorescence and fluorescein, optical crosstalk due to light scatter is minimized by the slightly opaque nature of our plate.

The use of centrifugal filtration allows the reliable collection of filtrate using the current AcroWell 96 filter plate configuration. Even without molded flow directors, centrifugal force directs the filtrate into the receiving plate without the risk of crosstalk. This feature, along with automation-friendly design, chemically resistant and low binding plate construction, and versatility, make AcroWell 96 filter plates the ideal tool to optimize sample preparation and detection assays.